Methods of treating parkinson&#39;s disease using viral vectors

ABSTRACT

Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson&#39;s Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/081,116, filedMar. 25, 2016, which is a continuation of U.S. patent application Ser.No. 13/647,155, filed Oct. 8, 2012, now U.S. Pat. No. 9,492,415, whichis a continuation of U.S. patent application Ser. No. 12/384,935, filedApr. 10, 2009, now U.S. Pat. No. 8,309,355, which is a divisional ofU.S. patent application Ser. No. 11/102,521, filed Apr. 8, 2005, nowU.S. Pat. No. 7,534,613, which is a continuation of U.S. patentapplication Ser. No. 09/887,854, filed Jun. 21, 2001, now U.S. Pat. No.6,953,575, which is a continuation of U.S. patent application Ser. No.09/320,171, filed May 26, 1999, now U.S. Pat. No. 6,309,634, which isrelated to U.S. Provisional Patent Application No. 60/134,748, filed May18, 1999, and U.S. Provisional Patent Application No. 60/086,949, filedMay 27, 1998, from which applications priority is claimed under 35 USC§§ 119/120, and which applications are incorporated herein by referencein their entireties.

REFERENCE TO GOVERNMENT RIGHTS

Part of this work was made under the auspices of the U.S. Department ofEnergy, at the University of California/Lawrence Berkeley NationalLaboratory, under CRADA No. BG98039 and DOE Contract No.DE-AC03-76SF00098, now Contract No. DE-AC02-05CH11231. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to efficient delivery of viralvectors to the CNS. More particularly, the present invention relates togene therapy for the treatment of central nervous system (CNS)disorders, particularly those disorders which involve theneurotransmitter dopamine.

BACKGROUND OF THE INVENTION

CNS disorders are major public health issues. Parkinson's disease (PD)alone affects over 1 million people in the United States. Clinically, PDis characterized by a decrease in spontaneous movements, gaitdifficulty, postural instability, rigidity and tremor. Parkinson'sdisease is caused by the degeneration of the pigmented neurons in thesubstantia nigra of the brain, resulting in decreased dopamineavailability. Altered dopamine metabolism has also been implicated inschizophrenic patients who show increased dopamine in certain areas ofthe brain. Currently, many CNS disorders such as PD are treated bysystemic administration of a therapeutic agent. Systemic administration,however, is often inefficient because of a drug's inability to passthrough the blood brain barrier and because many drugs cause peripheralside effects. Thus, many potentially useful compounds, such as proteins,cannot be administered systemically. If these compounds are successfulin penetrating the blood-brain-barrier, they may also induce centralnervous system side effects as well. Treatment of PD currently involvesoral administration of the dopamine-precursor, L-dopa often incombination with a compound such ascarbidopa, a peripheral inhibitor ofthe enzyme aromatic amino acid decarboxylase (AADC) that decarboxylatesdopa to dopamine. In the majority of patients, however, production ofAADC in the affected brain regions is reduced as PD progresses and,consequently, larger and larger doses of L-dopa are required, leavingthe patients with reduced therapeutic benefits and increased sideeffects.

In view of the limitations of current systemic therapies, gene deliveryis a promising method for the treatment for CNS disorders such as PD. Anumber of viral based systems for gene transfer purposes have beendescribed, such as retroviral systems which are currently the mostwidely used viral vector systems for this purpose. For descriptions ofvarious retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Millerand Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) HumanGene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns etal. (1993) Proc. Natl. Acad Sci. USA 90:8033-8037; and Boris-Lawrie andTemin (1993) Cur. Opin. Genet. Develop. 3:102-109.

Adeno-associated virus (AAV) systems are emerging as the leadingcandidates for use in gene therapy. AAV is a helper-dependent DNAparvovirus which belongs to the genus Dependovirus. AAV requiresinfection with an unrelated helper virus, either adenovirus, aherpesvirus or vaccinia, in order for a productive infection to occur.The helper virus supplies accessory functions that are necessary formost steps in AAV replication. For a review of AAV, see, e.g., Berns andBohenzky (1987) Advances in Virus Research (Academic Press, Inc.)32:243-307.

AAV infects a broad range of tissue, and has not elicited the cytotoxiceffects and adverse immune reactions in animal models that have beenseen with other viral vectors. (see, e.g., Muzyczka, (1992) CurrentTopics in Microbiol. and Immunol. 158:97-129; Flotte et al. (1993) PNASUSA 90:10613-10617; Kass-eiser et al. (1992) Gene Therapy 1:395-402;Yange et al. PNAS USA 91:4407-4411; Conrad et al. (1996) Gene Therapy3:658-668; Yang et al. (1996) Gene Therapy 3:137-144; Brynes et al.(1996) J. Neurosci. 16:3045-3055). Because it can transduce nondividingtissue, AAV may be well adapted for delivering genes to the centralnervous system (CNS). U.S. Pat. No. 5,677,158 described methods ofmaking AAV vectors. AAV vectors containing therapeutic genes under thecontrol of the cytomegalovirus (CMV) promoter have been shown totransduce mammalian brain and to have functional effects in models ofdisease.

AAV vectors carrying transgenes have been described, for example, inKaplitt et al. (1994) Nature Genetics 8:148-153; WO 95/28493 published26 Oct. 1995; WO 95/34670, published 21 Dec. 1995; During et al., (1998)Gene Therapy 5:820-827; Mandel et al. (1998) J. Neurosci. 18:4271-4284;Szczypka et al. (1999) Neuron 22:167-178.). However, delivery of AAVvectors to the CNS has proven difficult. AAV has been used to transferthe thymidine (tk) kinase gene to experimental gliomas in mice, and theability of AAV-tk to render these brain tumors sensitive to thecytocidal effects of ganciclovir has been demonstrated. Okada et al.(1996) Gene Therapy 3:959-964; Mizuno et al. (1998) Jpn. J. Cancer Res.89:76-80. Infusion of an AAV-CMV vector containing the human tyrosinehydroxylase (TH) gene, an enzyme involved in conversion of the aminoacid tyrosine to dopa, into adult rat brain resulted in transduction ofboth neurons and glia (Kaplitt et al. (1995) VIRAL VECTORS, GENE THERAPYAND NEUROSCIENCE APPLICATIONS, Kaplitt and Loewy eds., 12:193-210,Academic Press, San Diego; Bankiewicz et al. (1997) Exper. Neurol.144:147-156). Delivery of the same vector to monkey striatum resulted inrobust expression of TH for up to 2.5 months (During et al., supra).Furthermore, AAV-CMV-TH was tested in a rodent model of Parkinson'sDisease where it caused significant improvement in rotational behaviorof 6-hydroxydopamine-lesioned rats (Fan et al. (1998) Human Gene Therapy9:2527-2537; Mandel et al. (1997) PNAS USA 94:14083-14088).

However, while reports such as these demonstrate AAV's potential fortargeting the CNS, they also demonstrate that direct injection of AAVvectors into the CNS results in limited numbers of transfected cells andthat the transfected cells are clustered in a narrow area near theinjection tracts. (see, e.g., During et al, supra; Fan et al., supra).Since multiple injections into the CNS cause undesirable complications,there remains a need for methods of delivering AAV vectors to largerareas of the brain using the least number of injection sites. Inaddition, the relationship between dose of injected vector and itsresulting distribution in brain tissue has not been previously reported.

Furthermore, gene therapy of PD has focused on delivery of at least twogenes encoding enzymes involved in dopamine synthesis, namely TH andAADC. These methods are subject to all of the delivery problemsdiscussed above and, in addition, require that both genes are expressedin the proper amounts. Thus, treatment of PD using AAV-AADC incombination with L-dopa has also not been demonstrated.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for deliveringrecombinant AAV (rAAV) virions carrying a transgene to the centralnervous system (CNS) of a subject, for example a human, usingconvection-enhanced delivery (CED). CED can be conducted, for example,using either an osmotic pump or an infusion pump. In a preferredembodiment, the transgene encodes an aromatic amino acid decarboxylase(AADC) or active fragment thereof.

When the transgene encodes an AADC, it is preferable to administer therAAV virions into the striatum of the CNS.

In another aspect, the invention provides for methods for deliveringrecombinant AAV virions to a subject having a CNS disorder. The rAAVvirions encode a suitable therapeutic polypeptide and are administeredinto the CNS of the subject using CED. In a preferred embodiment, theCNS disorder is Parkinson's disease (PD), the rAAV virions areadministered into the striatum of the CNS, and the nucleic acid sequenceencodes AADC.

In another aspect, methods for treating a neurodegenerative disease in asubject are provided. A preparation of recombinant adeno-associatedvirus (rAAV) virions carrying a therapeutic nucleic acid sequence thatis expressible in transduced cells is administered to the CNS usingconvection-enhanced delivery (CED). In one embodiment, theneurodegenerative disease is PD and the therapeutic polypeptide is anAADC. In yet another embodiment, the method of treating theneurodegenative disease also includes administering at least oneadditional therapeutic compound to the subject, for example,systemically administering L-dopa and, optionally, carbidopa.

In yet another aspect, methods of determining levels of dopamineactivity in the CNS of subject are provided. A labeled tracer isadministered to the subject. The tracer is preferably a compound thatbinds to a cell which utilizes dopamine and the label is preferably aradioisotope, for instance, 6-[¹⁸F]-fluoro-L-m-tyrosine (¹⁸F-FMT). Thedetection of the label is indicative of dopamine activity via binding ofthe tracer. Preferably, the subject's CNS is imaged, for example usingpositron emission tomography (PET) scanning.

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1b, 1c and 1d , depict dose responses (expression of theAAV-tk transgene) in rat following intracranial infusion pump delivery.The tissue volume (FIG. 1a ); mean area (FIG. 1b ); length (FIG. 1c )and number of cells (FIG. 1d ) expressing the transgene are depicted.

FIG. 2 is a half-tone reproduction showing labeling of rat brain tissueafter injection of AAV vectors.

FIGS. 3a, 3b, 3c and 3d , depict intracranial delivery of AAV-tk througheither an infusion pump (IP) or osmotic pump (OP). The tissue volume(FIG. 3a ); mean area (FIG. 3b ); length (FIG. 3c ) and number of cells(FIG. 3d ) expressing the transgene are depicted.

FIGS. 4a, 4b, 4c and 4d are half-tone reproductions depicting CNS tissueinfused with with vector carrying the tk transgene. FIGS. 4a and b showexpression of tk in neurons. FIGS. 4c and d show expression in neuronsand glial near the site of osmotic pump infusion.

FIG. 5 is a half-tone reproduction depicting Southern blot analysis oftissues from a subject infused with AAV-tk vector.

FIG. 6 depicts immunostaining for AADC of the brains of MPTP-lesionedmonkeys. The left side (control) shows limited staining, while the rightside (AAV-AADC treated) shows broad AADC immunostaining.

FIG. 7 is an FMT PET scan depicting dopamine activity in the brains ofunilaterally MPTP-lesioned monkeys. The left side (baseline) showslimited activity on the lesioned side, while the right side (8 weekspost AAV-AADC administration) shows normal levels of dopamine activity.

FIGS. 8a, 8b and 8c , depict biochemical analysis of L-dopa levels inMPTP-lesioned monkeys. Panel A shows that L-dopa is converted todopamine by the AACD enzyme. In cortical regions, regardless of the MPTPtreatment, there is poor or no conversion of L-dopa to dopamine.Striatum is AADC-rich, therefore, most of the L-dopa has been convertedto dopamine in this region. One the striatum ipsilateral to MPTPadministration, L-dopa conversion to dopamine is impaired and similar tocortical activity in AAV-LacZ treated monkeys. Both AAV-AADC-treatedanimals show almost normal rates of L-dopa to dopamine conversion. PanelB depicts HVA analysis. HVA is a metabolite of dopamine catabolism.Since cortical regions are not able to convert L-dopa to dopamine HVAlevels are low. As shown in panel A, striatum converts L-dopa todopamine, therefore, dopamine is catabolised to HVA in this region.Since AADC activity has not been restored in the AAV-LacZ-treatedmonkeys HVA levels in MTPT ipsilateral striatum are low. HVA levels aresignificantly elevated in AAV-AADC-treated monkey in the MPTPipsilateral striatum. Panel C shows L-dopa levels were measured in thetissue punches following L-dopa administration. Due to the differentL-dopa absorption tissue levels differ between the monkeys. They aresimilar, however, within each subject. Tissue levels of L-dopa weredramatically reduced in the MPTP ipsilateral stratum of AAV-AADC-treatedmonkeys, since AADC enzyme has been restored. The activity of AADC inthis region is very strong, since tissue levels of L-dopa are lower thanin the contralateral striatum.

FIG. 9 is a graph depicting activity of the AADC enzyme in-vitro. Tissuepunches were incubated with L-dopa as described in the material andmethods. AADC enzyme activity was determined by measuring rates ofL-dopa to dopamine conversion. Cortical regions contain low levels ofAADC. AADC activity in contralateral striatum is high, however it isvariable since there is some dopaminergic lesion on that side of thebrain. AADC activity in MPTP ipsilatral striatum is significantlyreduced in AAV-Lac-Z-treated monkey while it is completely restored inthe AAV-DDC monkeys.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); CurrentProtocols in Molecular Biology (F. M. Ausubel, et al. eds., currentedition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.)

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting foreign DNA into host cells. Such methods can resultin transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene transfer provides a unique approachfor the treatment of acquired and inherited diseases. A number ofsystems have been developed for gene transfer into mammalian cells. See,e.g., U.S. Pat. No. 5,399,346.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a complete virus particle, such as a wild-type(wt) AAV virus particle (comprising a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

A “recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus composed of an AAV proteinshell, encapsidating a heterologous nucleotide sequence of interestwhich is flanked on both sides by AAV ITRs. A rAAV virion is produced ina suitable host cell which has had an AAV vector, AAV helper functionsand accessory functions introduced therein. In this manner, the hostcell is rendered capable of encoding AAV polypeptides that are requiredfor packaging the AAV vector (containing a recombinant nucleotidesequence of interest) into infectious recombinant virion particles forsubsequent gene delivery.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene1:197. Such techniques can be used to introduce one or more exogenousDNA moieties, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

The term “host cell” denotes, for example, microorganisms, yeast cells,insect cells, and mammalian cells, that can be, or have been, used asrecipients of an AAV helper construct, an AAV vector plasmid, anaccessory function vector, or other transfer DNA. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein generally refers to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

A “coding sequence” or a sequence which “encodes” a particular protein,is a nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom prokaryotic or eukaryotic mRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular polypeptide or proteinafter being transcribed or translated. Any of the polynucleotidesequences described herein may be used to identify larger fragments orfull-length coding sequences of the genes with which they areassociated. Methods of isolating larger fragment sequences are know tothose of skill in the art.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” conditionstypically hybridizes under conditions that allow detection of a targetnucleic acid sequence of at least about 10-14 nucleotides in lengthhaving at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.).

The term “aromatic amino acid decarboxylase” or “AADC” refers to apolypeptide which decarboxylates dopa to dopamine. Thus, the termincludes a full-length AADC polypeptide, active fragments or functionalhomologues thereof.

A “functional homologue,” or a “functional equivalent” of a givenpolypeptide includes molecules derived from the native polypeptidesequence, as well as recombinantly produced or chemically synthesizedpolypeptides which function in a manner similar to the referencemolecule to achieve a desired result. Thus, a functional homologue ofAADC encompasses derivatives and analogues of thosepolypeptides—including any single or multiple amino acid additions,substitutions and/or deletions occurring internally or at the amino orcarboxy termini thereof—so long as integrity of activity remains.

Techniques for determining nucleic acid and amino acid “sequenceidentity” or “homology” also are known in the art. Typically, suchtechniques include determining the nucleotide sequence of the mRNA for agene and/or determining the amino acid sequence encoded thereby, andcomparing these sequences to a second nucleotide or amino acid sequence.In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Two or more sequences(polynucleotide or amino acid) can be compared by determining their“percent identity.” The percent identity of two sequences, whethernucleic acid or amino acid sequences, is the number of exact matchesbetween two aligned sequences divided by the length of the shortersequences and multiplied by 100. An approximate alignment for nucleicacid sequences is provided by the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981). Thisalgorithm can be applied to amino acid sequences by using the scoringmatrix developed by Dayhoff, Atlas of Protein Sequences and Structure.M. O. Dayhoffed., 5 suppl. 3:353-358, National Biomedical ResearchFoundation, Washington, D.C., USA, and normalized by Gribskov, Nucl.Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of thisalgorithm to determine percent identity of a sequence is provided by theGenetics Computer Group (Madison, Wis.) in the “BestFit” utilityapplication. The default parameters for this method are described in theWisconsin Sequence Analysis Package Program Manual, Version 8 (1995)(available from Genetics Computer Group, Madison, Wis.). A preferredmethod of establishing percent identity in the context of the presentinvention is to use the MPSRCH package of programs copyrighted by theUniversity of Edinburgh, developed by John F. Collins and Shane S.Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide sequences are “substantially homologous” to eachother when the sequences exhibit at least about 80%-85%, preferably atleast about 90%, and most preferably at least about 95%-98% sequenceidentity over a defined length of the molecules, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

“Convection-enhanced delivery” refers to any non-manual delivery ofagents. In the context of the present invention, examples ofconvection-enhanced delivery (CED) of AAV can be achieved by infusionpumps or by osmotic pumps.

The term “central nervous system” or “CNS” includes all cells and tissueof the brain and spinal cord of a vertebrate. Thus, the term includes,but is not limited to, neuronal cells, glial cells, astrocytes,cereobrospinal fluid (CSF), interstitial spaces, bone, cartilage and thelike. The “cranial cavity” refers to the area underneath the skull(cranium). Regions of the CNS have been associated with variousbehaviors and/or functions. For example, the basal ganglia of the brainhas been associated with motor functions, particularly voluntarymovement. The basal ganglia is composed of six paired nuclei: thecaudate nucleus, the putamen, the globus pallidus (or pallidum), thenucleus accumbens, the subthalamic nucleus and the substantia nigra. Thecaudate nucleus and putamen, although separated by the internal capsula,share cytoarchitechtonic, chemical and physiologic properties and areoften referred to as the corpus striatum, or simply “the striatum.” Thesubstantia nigra, which degenerates in Parkinson's patients, providesmajor dopaminergic input into the basal ganglia.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, simians, humans, farm animals, sportanimals and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

The term “labeled tracer” refers to any molecule which can be used tofollow or detect a defined activity in vivo, for example, a preferredtracer is one that binds to cells that are utilizing dopamine.Preferably, the labeled tracer is one that can be viewed in a wholeanimal, for example, by positron emission tomograph (PET) scanning orother CNS imaging techniques.

Suitable labels include, but are not limited to radioisotopes,fluorochromes, chemiluminescent compounds, dyes, and proteins, includingenzymes.

General Overview of the Invention

Central to the present invention is the development of methods whichallow for the efficient delivery of viral vectors, such as AAV, into theCNS of animal. Previously, researchers have had only minimal successdelivering viral vectors to widespread areas of the brain. Usingconvection-enhanced delivery devices (for example, osmotic or infusionpumps), viral vectors can be delivered to many cells over large areas ofthe brain. Moreover, the delivered vectors efficiently expresstransgenes in CNS cells (e.g., neurons or glial cells).

Using the methods of viral vector delivery described herein, novel genetherapy treatments for CNS disorders (e.g., Parkinson's Disease) can bedevised. In one embodiment, Parkinson's disease (PD) is treated bycombining systemic L-dopa and/or carbidopa therapy withCNS-administration (e.g., via CED) of AAV vectors carrying a transgeneencoding AADC, an enzyme involved in dopamine metabolism.

Advantages of the invention, include, but are not limited to (i)efficient and widespread delivery of viral vectors (such as AAV) to theCNS; (ii) expression of nucleic acids (e.g., transgenes) carried by theviral vectors; (iii) identification of a therapeutic regime forParkinson's Disease that involves delivery of one transgene incombination with administration of a pro-drug; and (iv) the ability tonon-invasively monitor CNS gene therapy using PET scan.

Construction of Viral Vectors

Gene delivery vehicles useful in the practice of the present inventioncan be constructed utilizing methodologies well known in the art ofmolecular biology (see, for example, Ausubel or Maniatis, supra).Typically, viral vectors carrying transgenes are assembled frompolynuclotides encoding the transgene(s), suitable regulatory elementsand elements necessary for production of viral proteins which mediatecell transduction. For example, in a preferred embodiment,adeno-associated viral (AAV) vectors are employed.

General Methods

A preferred method of obtaining the nucleotide components of the viralvector is PCR. General procedures for PCR are taught in MacPherson etal., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford University Press,(1991)). PCR conditions for each application reaction may be empiricallydetermined. A number of parameters influence the success of a reaction.Among these parameters are annealing temperature and time, extensiontime, Mg²⁺ and ATP concentration, pH, and the relative concentration ofprimers, templates and deoxyribonucleotides. Exemplary primers aredescribed below in the Examples. After amplification, the resultingfragments can be detected by agarose gel electrophoresis followed byvisualization with ethidium bromide staining and ultravioletillumination.

Another method for obtaining polynucleotides is by enzymatic digestion.For example, nucleotide sequences can be generated by digestion ofappropriate vectors with suitable recognition restriction enzymes. Theresulting fragments can then be ligated together as appropriate.

Polynucleotides are inserted into vector genomes using methods wellknown in the art. For example, insert and vector DNA can be contacted,under suitable conditions, with a restriction enzyme to createcomplementary or blunt ends on each molecule that can pair with eachother and be joined with a ligase. Alternatively, synthetic nucleic acidlinkers can be ligated to the termini of a polynucleotide. Thesesynthetic linkers can contain nucleic acid sequences that correspond toa particular restriction site in the vector DNA. Other means are knownand available in the art.

Retroviral and Adenoviral Vectors

A number of viral based systems have been used for gene delivery. Forexample, retroviral systems are known and generally employ packaginglines which have an integrated defective provirus (the “helper”) thatexpresses all of the genes of the virus but cannot package its owngenome due to a deletion of the packaging signal, known as the psisequence. Thus, the cell line produces empty viral shells. Producerlines can be derived from the packaging lines which, in addition to thehelper, contain a viral vector which includes sequences required in cisfor replication and packaging of the virus, known as the long terminalrepeats (LTRs). The gene of interest can be inserted in the vector andpackaged in the viral shells synthesized by the retroviral helper. Therecombinant virus can then be isolated and delivered to a subject. (See,e.g., U.S. Pat. No. 5,219,740.) Representative retroviral vectorsinclude but are not limited to vectors such as the LHL, N2, LNSAL, LSHLand LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740,incorporated herein by reference in its entirety, as well as derivativesof these vectors, such as the modified N2 vector described herein.Retroviral vectors can be constructed using techniques well known in theart. See, e.g., U.S. Pat. No. 5,219,740; Mann et al. (1983) Cell:153-159.

Adenovirus based systems have been developed for gene delivery and aresuitable for delivery according to the methods described herein. Humanadenoviruses are double-stranded DNA viruses which enter cells byreceptor-mediated endocytosis. These viruses are particularly wellsuited for gene transfer because they are easy to grow and manipulateand they exhibit a broad host range in vivo and in vitro. For example,adenoviruses can infect human cells of hematopoietic, lymphoid andmyeloid origin. Furthermore, adenoviruses infect quiescent as well asreplicating target cells. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis. The virus is easilyproduced at high titers and is stable so that it can be purified andstored. Even in the replication-competent form, adenoviruses cause onlylow level morbidity and are not associated with human malignancies.Accordingly, adenovirus vectors have been developed which make use ofthese advantages. For a description of adenovirus vectors and their usessee, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.(1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human GeneTherapy 6:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al.(1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476.

AAV Expression Vectors

In a preferred embodiment, the viral vectors are AAV vectors. By an “AAVvector” is meant a vector derived from an adeno-associated virusserotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or part, preferably the rep and/or cap genes, butretain functional flanking ITR sequences. Functional ITR sequences arenecessary for the rescue, replication and packaging of the AAV virion.Thus, an AAV vector is defined herein to include at least thosesequences required in cis for replication and packaging (e.g.,functional ITRs) of the virus. The ITRs need not be the wild-typenucleotide sequences, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides, so long as the sequencesprovide for functional rescue, replication and packaging.

AAV expression vectors are constructed using known techniques to atleast provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an“AAV ITR” need not have the wild-type nucleotide sequence depicted, butmay be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

Additionally, AAV ITRs may be derived from any of several AAV serotypes,including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7,etc. Furthermore, 5′ and 3′ ITRs which flank a selected nucleotidesequence in an AAV expression vector need not necessarily be identicalor derived from the same AAV serotype or isolate, so long as theyfunction as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

Suitable DNA molecules for use in AAV vectors will be less than about 5kilobases (kb) in size and will include, for example, a gene thatencodes a protein that is defective or missing from a recipient subjector a gene that encodes a protein having a desired biological ortherapeutic effect (e.g., an antibacterial, antiviral or antitumorfunction). Preferred DNA molecules include those involved in dopaminemetabolism, for example, AADC or TH. AAV-AADC and AAV-TH vectors havebeen described, for example, in Bankiewicz et al. (1997) Exper't Neurol.144:147-156; Fan et al (1998) Human Gene Therapy 9:2527-2535 andInternational Publication WO 95/28493, published Oct. 26, 1995.

The selected nucleotide sequence, such as AADC or another gene ofinterest, is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, the SV40 early promoter, mouse mammarytumor virus LTR promoter; adenovirus major late promoter (Ad MLP); aherpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, synthetic promoters, hybrid promoters, and thelike. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, will also find use herein. Such promotersequences are commercially available from, e.g., Stratagene (San Diego,Calif.).

For purposes of the present invention, both heterologous promoters andother control elements, such as CNS-specific and inducible promoters,enhancers and the like, will be of particular use. Examples ofheterologous promoters include the CMB promoter. Examples ofCNS-specific promoters include those isolated from the genes from myelinbasic protein (MBP), glial fibrillary acid protein (GFAP), and neuronspecific enolase (NSE). Examples of inducible promoters include DNAresponsive elements for ecdysone, tetracycline, hypoxia and aufin.

The AAV expression vector which harbors the DNA molecule of interestbounded by AAV ITRs, can be constructed by directly inserting theselected sequence(s) into an AAV genome which has had the major AAV openreading frames (“ORFs”) excised therefrom. Other portions of the AAVgenome can also be deleted, so long as a sufficient portion of the ITRsremain to allow for replication and packaging functions. Such constructscan be designed using techniques well known in the art. See, e.g., U.S.Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar.1993); Lebkowski et al. (1988) Molec. Cell. Biol. 1:3988-3996; Vincentet al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter,B. J. (1992) Current Opinion in Biotechnology 2:533-539; Muzyczka, N.(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R.M. (1994) Human Gene Therapy 1:793-801; Shelling and Smith (1994) GeneTherapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 ug/ml BSA, 10 mM-50 mM NaCl, and either 40 uM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0 □C (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14□C (for“blunt end” ligation). Intermolecular “sticky end” ligations are usuallyperformed at 30-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). AAV vectors which contain ITRs have been described in,e.g., U.S. Pat. No. 5,139,941. In particular, several AAV vectors aredescribed therein which are available from the American Type CultureCollection (“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 12:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad Sci.USA 4:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

For the purposes of the invention, suitable host cells for producingrAAV virions include microorganisms, yeast cells, insect cells, andmammalian cells, that can be, or have been, used as recipients of aheterologous DNA molecule. The term includes the progeny of the originalcell which has been transfected. Thus, a “host cell” as used hereingenerally refers to a cell which has been transfected with an exogenousDNA sequence. Cells from the stable human cell line, 293 (readilyavailable through, e.g., the American Type Culture Collection underAccession Number ATCC CRL1573) are preferred in the practice of thepresent invention. Particularly, the human cell line 293 is a humanembryonic kidney cell line that has been transformed with adenovirustype-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), andexpresses the adenoviral E1a and E1b genes (Aiello et al. (1979)Virology 94:460). The 293 cell line is readily transfected, and providesa particularly convenient platform in which to produce rAAV virions.

AAV Helper Functions

Host cells containing the above-described AAV expression vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virions. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV expression vectors. Thus, AAV helperfunctions include one, or both of the major AAV ORFs, namely the rep andcap coding regions, or functional homologues thereof.

The Rep expression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves.

AAV helper constructs can be in the form of a plasmid, phage,transposon, cosmid, virus, or virion. A number of AAV helper constructshave been described, such as the commonly used plasmids pAAV/Ad andpIM29+45 which encode both Rep and Cap expression products. See, e.g.,Samulski et al. (1989)J. Virol. 63:3822-3828; and McCarty et al. (1991)J. Virol. 65:2936-2945. A number of other vectors have been describedwhich encode Rep and/or Cap expression products. See, e.g., U.S. Pat.No. 5,139,941.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 1:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

Both AAV expression vectors and AAV helper constructs can be constructedto contain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to G418 (available from Sigma,St. Louis, Mo.). Other suitable markers are known to those of skill inthe art.

AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding non AAV derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are non AAV derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

Particularly, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon orcosmid. Accessory vectors can also be in the form of one or morelinearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions. See, forexample, International Publication No. WO 97/17548, published May 15,1997.

Nucleic acid sequences providing the accessory functions can be obtainedfrom natural sources, such as from the genome of an adenovirus particle,or constructed using recombinant or synthetic methods known in the art.In this regard, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized. See, e.g.,Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRCHandbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N.(1992) Curr. Topics. Microbiol. and Immun. 158:97-129. Specifically,early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1bare thought to participate in the accessory process. Janik et al. (1981)Proc. Natl. Acad Sci. USA 78:1925-1929. Herpesvirus-derived accessoryfunctions have been described. See, e.g., Young et al. (1979) Prog. MedVirol. 25:113. Vaccinia virus-derived accessory functions have also beendescribed. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al.(1986) Virology 152:110-117.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest) fromthe AAV expression vector. The Rep proteins also serve to duplicate theAAV genome. The expressed Cap proteins assemble into capsids, and therecombinant AAV genome is packaged into the capsids. Thus, productiveAAV replication ensues, and the DNA is packaged into rAAV virions.

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas CsCl gradients. Further, if infection is employed to express theaccessory functions, residual helper virus can be inactivated, usingknown methods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60□C for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions are then ready for use for DNA delivery tothe CNS (e.g., cranial cavity) of the subject.

Delivery of Viral Vectors

Methods of delivery of viral vectors include, but are not limited to,intra-arterial, intra-muscular, intravenous, intranasal and oral routes.Generally, rAAV virions may be introduced into cells of the CNS usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

For in vivo delivery, the rAAV virions will be formulated intopharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

However, since conventional methods such as injection have not beenshown to provide widespread delivery of viral vectors to the brain ofthe subject, central to the present invention is the discovery thatviral vectors are efficiently delivered to the CNS viaconvection-enhanced delivery (CED) systems. The present inventors arethe first to describe and demonstrate that CED can efficiently deliverviral vectors, e.g., AAV, over large regions of an animal's brain (e.g.,striatum). As described in detail and exemplified below, these methodsare suitable for a variety of viral vectors, for instance AAV vectorscarrying reporter genes (e.g., thymidine kinase (tk)) or therapeuticgenes (e.g., AADC and tk).

Any convection-enhanced delivery device may be appropriate for deliveryof viral vectors. In a preferred embodiment, the device is an osmoticpump or an infusion pump. Both osmotic and infusion pumps arecommerically available from a variety of suppliers, for example AlzetCorporation, Hamilton Corporation, Alza, Inc., Palo Alto, Calif.).Typically, a viral vector is delivered via CED devices as follows. Acatheter, cannula or other injection device is inserted into CNS tissuein the chosen subject. In view of the teachings herein, one of skill inthe art could readily determine which general area of the CNS is anappropriate target. For example, when delivering AAV-AADC to treat PD,the striatum is a suitable area of the brain to target. Stereotacticmaps and positioning devices are available, for example from ASIInstruments, Warren, Mich. Positioning may also be conducted by usinganatomical maps obtained by CT and/or MRI imaging of the subject's brainto help guide the injection device to the chosen target. Moreover,because the methods described herein can be practiced such thatrelatively large areas of the brain take up the viral vectors, fewerinfusion cannula are needed. Since surgical complications are related tothe number of penetrations, the methods described herein also serve toreduce the side effects seen with conventional delivery techniques.

Pharmaceutical compositions will comprise sufficient genetic material toproduce a therapeutically effective amount of the protein of interest,i.e., an amount sufficient to reduce or ameliorate symptoms of thedisease state in question or an amount sufficient to confer the desiredbenefit. The pharmaceutical compositions will also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, sorbitol,Tween80, and liquids such as water, saline, glycerol and ethanol.Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present invention becombined with other suitable compositions and therapies. For instance,as described in detail in the Examples below, Parkinson's disease can betreated by co-administering an AAV vector expressing AADC into the CNS(e.g., into the caudate nucleus or putamen of the striatum) andadditional agents, such as dopamine precursors (e.g., L-dopa),inhibitors of dopamine synthesis (e.g., carbidopa), inhibitors ofdopamine catabolism (e.g., MaOB inhibitors), dopamine agonists orantagonists can be administered prior or subsequent to or simultaneouslywith the vector encoding AADC. For example, L-dopa and, optionally,carbidopa, may be administered systemically. In this way, the dopaminewhich is naturally depleted in PD patients, is restored, apparently byexpression of AADC which is able to convert L-dopa into dopamine. Wherethe transgene (e.g., AADC) is under the control of an induciblepromoter, certain systemically-delivered compounds such as muristerone,ponasteron, tetracyline or aufin may be administered in order toregulate expression of the transgene.

Treatment of CNS Disorders

Viral vectors expressing therapeutic transgenes can be used to treatvarious CNS disorders by providing therapeutic proteins or polypeptides.In a preferred embodiment, the viral vectors are delivered to the CNSvia the CED methods described herein as these methods provide the firsteffective way of broadly distributing viral vectors into the CNS.Non-limiting examples of disorders which may be treated include tumors,injury resulting from stroke and neurodegenerative diseases.

Parkinson's Disease

In a preferred embodiment of the present invention, viral vectors whichprovide the enzyme AADC are used for the treatment of Parkinson'sdisease. As described above, Parkinson's disease results from aselective loss of dopaminergic nigrostriatal neurons, resulting in aloss of input from the substantia nigra to the striatum. Animal modelsof PD have been created, for instance by treating rats or primates with6-hydroxydopamine (6-OHDA) to destroy dopaminergic cells or by lesioningprimates with the neurotoxin1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), which produces aParkinson's-like disease.

The present invention provides the first evidence that dopaminergicactivity can be restored in Parkinson's patients (e.g., MPTP-lesionedmonkeys), by administration of viral vectors carrying the transgene forAADC in combination with systemic (e.g., oral) administration of L-dopaand, optionally, carbidopa. Previous suggestions for gene therapy forParkinson's have focused on the deficiency of tyrosine hydroxylase asthe disease progresses. These suggestions, therefore, call for therestoration of dopamine synthesis in the nigrostriatal pathway by thesuccessful expression of at least three transgenes in order to makedopamine in situ: the tyrosine hydroxylase gene; the gene for theco-factor bioptrene, GTP-cyclohydroxylase-1; and the gene for AADC. Inaddition to the problems associated with delivering three genes atappropriate levels, the regulation of dopamine levels would be difficultto control using this approach.

The present inventors have demonstrated that one transgene (e.g., AADC)in combination with L-dopa provides therapeutic benefit. AADC is theenzyme involved in the final step of dopamine biosynthesis, convertingL-dopa to dopamine. Thus, a clear advantage of the AADC therapeuticapproach to restoring dopaminergic activity is that only one gene has tobe delivered and the regulation of dopamine levels is possible bycontrolling peripheral levels of L-dopa. Furthermore, by delivering justthe AADC gene, L-dopa can be used as a pro-drug to regulate levels ofdopamine in the striatum.

Since AADC-encoding nucleotides delivered by AAV vectors appear to beexpressed mainly in the striatal neurons another important therapeuticadvantage is the treatment's provision of a buffering mechanism forL-dopa. Many side effects, such as dyskinesisas, are attributed to theinefficient buffering of Parkinsonian brain. The methods describedherein avoid this problem by allowing un-metabolized L-dopa to be storedin the neurons. As exemplified below, the delivery of AADC to theMPTP-treated striatum enables conversion of L-dopa to dopamine and thesubsequent metabolism to DOPAC and HVA by striatal neurons. Based on FMTPET data, it appears that striatal neurons can also store dopamine,since FMT was visualized in this region. In fact, conversion rates ofL-dopa to dopamine following AADC gene transfer were as robust andgreater than seen in the normal striatum (see, e.g., FIG. 7).Furthermore, although Parkinson's disease is an progressive disorder, itis not likely that an ongoing degeneration process will affect AADCexpression in striatal neurons since they are not typically affected byidopathic Parkinson's disease.

The degeneration of the dopaminergic system in patients with idiopathicParkinson's disease is not uniform. The nigrostriatal pathwaydegenerates at much faster rate than mesolimbic pathway, leavingpatients with an imbalance between the activity of the two pathways. Asthe disease progresses, higher levels of L-dopa are needed to compensatefor the degeneration of the nigrostriatal pathway, but this also resultsin increasingly higher dopamine levels in the nucleus accumbens andother parts of the mesolimbic system. Such overstimulation may beresponsible for some of the side effects associated with L-dopatreatment such as hallucinations. Similarly, MPTP leaves the mesolimbicdopaminergic system relatively spared (see, FIG. 7, no dopaminergicinnervation is present in the caudate and putamen, a partial lesion isseen in the nucleus accumbens). As shown in FIG. 7, AAV-AADC can restorethis imbalance almost back to normal, therefore, it is possible thatlower doses of L-dopa will be required following the restoration of AADCenzyme levels and improved L-dopa to dopamine conversion rates. This inturn might reduce overstimulation of the mesolimbic system, resulting infewer L-dopa/carbidopa related side effects.

As explained above, the AAV-AADC vectors can be delivered by anysuitable method, for example, injection, grafting, infusion,transplantation of cells carrying the vectors, etc. In a preferredembodiment, the vectors are delivered by the CED methods describedherein. As exemplified below, such delivery methods provide broaddistribution and expression in CNS neurons and thereby provide a noveltreatment regime for PD.

Imaging

The present invention also provides methods of determining in vivoactivity of an enzyme or other molecule. More specifically, a tracerwhich specifically tracks the targeted activity is selected and labeled.In a preferred embodiment, the tracer tracks dopamine activity, forexample fluoro-L-m-tyrosine (FMT) which binds to cells that utilizedopamine. Suitable labels for the selected tracer include anycomposition detectable by spectroscopic, photochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include radiolabels (e.g., ¹⁸F, ³H, ¹²⁵I, ³⁵S, ³²P, etc),enzymes, colorimeteric labels, fluorescent dyes, and the like. In apreferred embodiment, the label ¹⁸F is used with FMT to quantifydopamine activity.

Means of detecting labels are well know to those of skill in the art.For example, radiolabels may be detected using imaging techniques,photographic film or scintillation counters. In a preferred embodiment,the label is detected in vivo in the brain of the subject by imagingtechniques, for example positron emission tomography (PET). PETtechniques are discussed in detail in Example 3 below.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1: Construction and Production of AAV-TK

The AAV-tk vector was constructed by placing the herpes simplex virusthymidine kinase (tk) gene under the transcriptional control of thecytomegalovirus (CMV) immediate early promoter in a pUC-based plasmid(available from, Roche Molecular Biochemicals). A β-globin intron waslocated directly upstream from the tk gene and human growth hormonepoly-A was placed downstream. The entire cassette was flanked by AAVinverted terminal repeats (ITRs) that are required for gene expression,replication, and packaging into viral particles.

Recombinant AAV virions were produced in human 293 cells (readilyavailable through, e.g., the American Type Culture Collection underAccession Number ATCC CRL1573) as follows. The 293 cell line wascultured in complete DMEM (Biowhittaker) containing 4.5 g/liter glucose,10% heat-inactivated fetal calf serum (FCS; Hyclone), and 2 mMglutamine. Subconfluent 293 cells were co-transfected by calciumphosphate precipitation (see, e.g., Sambrook, et al.) with the AAV-tkexpression cassette flanked by ITRs and helper plasmids derived fromboth AAV (pw1909, containing the AAV rep and cap genes) and adenovirus(pLadenol, containing E2a, E4, and adenoviral VA₁ and VA₁₁ RNA genes).After 6 hours, the media was changed to DMEM without serum andincubation was continued at 37° C. in 5% CO₂ for 72 hours. Pelletedcells were lysed in Tris buffer (100 mM Tris/150 mM NaCl, pH 8-0) bythree cycles of freeze/thaw, and lysate was clarified of cell debris bycentrifugation at 10,000 g for 15 m. To pellet non-viral proteins, theclarified lysate was centrifuged at 10,000 g for 15 min after addingCaCl₂ to a final concentration of 25 mM and incubated for 1 h at 0 C.Polyethylene glycol 8000 (PEG) was added to the resulting supernatant(final concentration=8%); this solution was incubated for 3 h at 0° C.and centrifuged at 3000×g for 30 minutes. The vector-containing pelletwas solubilized in 50 mM Hepes Na/150 mM NaCl/25 mM EDTA, pH 8.0, andcentrifuged at 10,000×g for 15 minutes to pellet and remove insolublematerial.

Cesium chloride isopycnic gradient centrifugation was performed andAAV-tk was recovered from the resulting gradient by isolating thefractions with in average density of 1.38 g/ml. PEG was again used toconcentrate vector, which was then resuspended in 25 mM Hepes Na/150 mMNaCl, pH 7.4 and centrifuged as described to remove insoluble material.The stock was treated with DNAse and vector titer was determined byquantitative dot-blot hybridization.

Example 2: In Vivo Delivery of AAV-TK: Dosages and Methods

In order to determine the appropriate dose of AAV to introduce into thebrain, the following study was conducted. The striatum was used to testdose response to the AAV vector because of its relatively large area ofhomogenous tissue and because it is a target for treatment ofneurodegenerative disease and other central nervous system disorders.

In addition, efficient methods of delivering vector to the CNS weredetermined. Simple stereotactic injection of therapeutic agents has beenshown to result in limited volume of distribution in brain (Kroll et al,(1996) Neurosurgery 38:746-754). Therefore, slow infusion pumps wereused to maintain a pressure gradient during intracranial delivery.Previous studies concerned with the delivery of small, medium, and largemolecules to brain have demonstrated that slow infusion pump results inextensive and homogenous tissue distribution.

To investigate which method of administering intracranial injection ofthe vector is most efficient, rats were given 2.5×10¹⁰ particles ofAAV-tk by using the Harvard infusion pump (Harvard Apparatus Inc.,Holliston, Mass.) or Alzet subcutaneous osmotic pumps (Alza ScientificProducts, Palo Alto, Calif.). Female Sprague-Dawley rats (250-300 g)from Charles River Laboratories (Wilmington, Mass.) were anesthetizedwith an intraperitoneal injection of ketamine (100 mg/kg body weight)and xylazine (10 mg/kg, body weight) and prepped for surgery. Duringsurgery, sedation was maintained with isofluorane (Attrane, Omeda PPDInc., Liberty, N.J.) and O₂ flow rates were kept at 0.3-0.5 L/m. Thehead of each rat was fixed in a stereotactic apparatus (Small AnimalStereotactic Frame; ASI Instruemnts, Warren, Mich.) with ear bars, and amidline incision was made through the skin to expose the cranium. A borehole was made in the skull 1 mm anterior to the bregma and 2.6 mmlateral to the midline using a small dental drill. Vector was deliveredto the left hemisphere and a depth of 5 mm using an infusion pump orsubcutaneous osmotic pumps.

For dosage studies, there were 3 groups of animals with 6 animals pergroup. AAV-tk was continually administered to each rat at a rate of 8μl/h for 2.5 h using a Harvard infusion pump. The loading chamber(Teflon tubing 1/16th″ OD×0.03″ ID) and attached infusion chamber (1/16″ OD×0.02″ ID) were filled with 2.5×10⁸, 2.5×10⁹, or 2.5×10¹⁰particles of AAV-tk in a total volume of 20 μl artificial csf (148 mMNaCl, 3 mM KCl, 1.4 mM CaCl₂*2H₂O, 0-8 mM MgC₂.6H₂O, 1.3 mM Na₂HPO₄*H₂O,0.2 mM Na₂HPO₄.H₂O). Delivery was through a 27 gauge needle fitted withfused silica, which was gradually removed 15 m following infusion.

Alternatively, subcutaneous osmotic pumps were used to deliver vector toone group of 6 animals. AAV-tk was continually administered to each ratat a rate of 8 μl/hour for 24 h using Alzet osmotic pumps, model #2001D(ALZA Scientific Products, Palo Alto, Calif.). The pump's reservoir andattached catheter (polyethylene 60 tubing) were filled with 2.5×10¹⁰particles of AAV-tk in a total volume of 200 μl artificial csf (HarvardApparatus, Inc., Holliston, Mass.) Delivery was through a 27 gaugecannula fitted with fused silica. After stereotactic placement, thecannula was secured to the skull with a small stainless steel screw anddental cement, and the pump was implanted subcutaneously in themid-scapular area of the back. The surgical site was closed inanatomical layers with 9 mm wound clips. Twenty four hours later, pumpswere removed by clipping and sealing the catheters but the implantedcannulas were left in place. Burr holes were filled with bone wax.

All surgical procedures, animal care and housing, and tissue harvestwere performed at the Richmond facility of the Berkeley Antibody Co.(Berkeley, Calif.).

Histology

Animals were euthanized by pentobarbitol overdose (dose) and perfusedthrough the ascending aorta with ice-cold PBS and 4% neutral bufferedparaformaldehyde. The brains were removed from the skull, post-fixed byimmersion in the same fixative for 24 hr, equilibrated in 30% sucrose,and frozen in −70° C. isopentane. They were then positioned in acryostat and 40 micron sections were serially collected from theprefrontal cortex to the midbrain. Each brain yielded approximately 150sections, that spanned an anterior-posterior (AP) length of 6-8 mm. Forroutine histological analysis, every IP section was stained with H & E.Selected sections representing different brain regions were stained withcrystal violet in order to determine general cell density. Results areshown in Table 1.

TABLE 1 Cellular Infilt. Necrosis Bleeding Fresh Hemosiderin TrackNeedle Infusion Pump − − − − Fine lines (2 × 10⁸) Infusion Pump − − + −Fine lines (2 × 10⁹) Infusion Pump − ++ ++ − Fine lines (2 × 10¹⁰) (1/6)(4/6) Osmotic Pump +++ +++ ++ +++ Holes (2 × 10¹⁰) (5/6)

PCR Analysis

Two additional groups of rats, with two animals per group, were treatedwith 2.5×10¹⁰ particles AAV-tk for the purpose of determining tissuedistribution of vector. One of the groups received vector by infusionand the other by osmotic pump, as described above. Animals wereeuthanized three weeks later using CO₂ inhalation and samples from 15organs and tissues were harvested from each rat including right brain,left brain, spinal cord, right eye, left eye, heart, lung, liver,kidney, spleen, ovary, thymus, lymph node, bone marrow, and leg muscle.Sterile techniques were used and tissue was collected using disposablesuture removal kits that were changed between each sample. Tissues wereimmediately frozen in liquid N₂ and kept at −70° C. until they wereprocessed for genomic DNA. PCR was performed using Perkin Elmer'sGeneAmp PCR Core Kit and two 30-mer oligos derived from tk sequence(5′-AAGTCATCGGCTCGGGTACGTAGACGATATC-3′ (SEQ ID NO:1) and 5′ATAGCAGCTACAATCCAGCTACCATTCTGC-3′ (SEQ ID NO:2)). Reactions wereperformed in a PTC-100 thermal cycler (MJ Reserach, Inc.) and resultedin a 158 bp per product in samples where vector was present.

Immunohistochemisty

Immunocytochemistry was used to detect transgene expression in everysection that directly followed one stained with H & E. Thus, one out ofevery 12 sections was washed in PBS, treated with 3% H₂O₂ for 30 m toblock endogenous peroxidase activity, rinsed again in dH₂O and PBS, andincubated in blocking solution (10% goat serum+0.01% Triton-X100 in PBS)for 30 m. Next, samples were incubated in polygonal anti-tk antibody(Yale) (1:1000) for 1 h, washed three times in PBS, incubated inbiotinylated goat anti-rabbit IgG (Vector) (1:300) for 1 h, and washedagain. Antibody binding was visualized with Streptavidin horseradishperoxidase (1:300) and VIP chromogen (Vector).

Quantitative Analysis

Transgene expression was quantitated for each brain by using aKen-a-vision microprojector to project tk-immunostained sections onto anARTZII graphic tablet. The NIH image 1.6 program was used to capture andanalyze images. The total estimated number of positive cells for eachbrain was determined at a magnification of 100× using the followingformula:

Total tk cell #=(n1+n2+n3 . . . )×12×k

n=positive cells/section

k: correction factor derived from the Ambercrombie equation (1946):

k=T/(T+D) T=section thickness (40μ), D cell diameter (16); k=0.71

The volumes of the tk-immunoreactive regions were determined bymeasuring areas of tk expression using captured images projected at 50×magnification and calculating as follows:

     V = A_(x) × L$\mspace{76mu} {A_{x} = {{{average}\mspace{14mu} {area}\mspace{14mu} \left( {mm}^{2} \right)} = \frac{{a\; 1} + {a\; 2} + {a\; 3\ldots \; {An}}}{n}}}$(where, L = AP  distance  (μ)  of  staining = n × 12 × 40u  and  n = #  of  stained  sections)

To determine how much virus is required to efficiently transduce braintissue, comparisons of immunostained sections were performed betweenrats receiving 2.5×10⁸, 2.5×10⁹, or 2.5×10¹⁰ particles of AAV-tk via theHarvard infusion pump.

With all parameters measured, a clear dose response was observed.Infused tissue from animals receiving the highest titer demonstratedtransgene expression in an average of 300 mm³ of tissue (orapproximately 60% of an adult rat cerebral hemisphere (Leyden et al.(1998) Behav. Brain Res. 87:59-67) as compared to volumes of 10 mm³ forthe middle-dose group and <1 mm³ for the low-dose group (FIG. 1a ).Volumes were calculated from the mean areas and lengths of stainingwhich both also showed significant differences between the groups (FIG.1 b,c). Expression within a volume of transduced tissue was not uniform,however, but exhibited a gradient of staining. Areas directlysurrounding the injection sites were heavily labeled while fewerpositive cells could be detected as distance from the needle tractincreased (FIG. 2). Finally, FIG. 1d illustrates that the total numberof tk-positive cells in section, from the high dose group, estimated toaverage 169,000, is approximately 10× higher than that of the middledose group.

Infusion Versus Osmotic Pump Delivery

Comparisons of immunostained brain sections demonstrated similaritiesand differences in the abilities of the two pumps to deliver vector.There was no significant difference in tk expression between the twogroups as measured by mean volume, area, AP distance, and totalestimated number of positive cells (FIG. 3a-d ). Because there was sometissue loss surrounding the needle tracts of all of the samples withinthe osmotic pump delivery group (data not shown), that group's true meanvalue for the number of estimated positive cells may be higher. And,while both delivery methods resulted in notable transgene expression,there was a difference in the type of cells that became labeled. Tissueinfused with vector expressed tk almost exclusively in neurons (FIG. 4ab) and tissue receiving vector via osmotic pump exhibited expression inneurons and in reactive glial cells close to the site of injection (FIG.4c,d ).

Tissue Distribution

To determine if recombinant AAV could be detected at locations distantfrom the site of intracranial delivery, PCR analysis was performed on 15different organs and tissue from each of three rats who had received ahigh dose of vector. Regardless of the delivery method (infusion orosmotic pumps), a 458 bp PCR product from the tk gene could be detectedin spinal cord, spleen, and both hemispheres of the brain using Southernblot analysis (FIG. 6). In one of the rats, vector sets were alsodetected in tissue from the kidney.

Toxicity

To assess whether or not toxicity was associated with any of thedelivery methods, histopathology was performed on H & E sections fromeach group and the results are summarized in table 1. Overall tissuemorphology was well preserved and no freezing or other artifacts werepresent. In the infusion delivery groups, tissue damage was minimal, ifpresent at all. There was no cellular infiltration, no necrosis in theneedle tract, and minimal cortical necrosis in a few of the animals.Fresh bleeding was found in one of the high-dose rats, andhemosiderosis, indicating moderate bleeding in the past, was found infour of the high-dose animals. Alternatively, serious damage was notedin all of the animals in the osmotic pump delivery group including largenecrotic areas surrounding the needle tract, cellular infiltrates, andhemosiderosis.

Thus, infusion of 2.5×10¹⁰ particles of AAV-tk at 8 μl/h for 2.5 h issufficient to partially distribute AAV vector to a volume of 300 mm³ oftissue. Within this region, a gradient of expression is observed withheavy staining directly surrounding the site of injection and fewerpositive cells farther away. Distribution appears to be a function ofdose (particle number) and not a function of delivery time or samplevolume when two different pump delivery systems are compared: thedistribution of 2.5×10¹⁰ particles was the same whether it was deliveredby osmotic pump (volume=200 μl, rate=8 μl/h, time=24 h) or infusion pump(volume=20 μl, rate=8 μl/h, time=2.5 h). Furthermore, strikinglydifferent levels of expression were observed between the three infusiondelivery groups, where sample volume, rate, and delivery time were keptconstant and particle number was the only variable.

These results obtained with convection-enhanced delivery of AAV areconsistent with those obtained from CED studies of large macromolecules,such as supramagnetic particles to rat brains. (Kroll et al. (1996)Neurosurg. 38:746-754, U.S. Pat. No. 5,720,720). Kroll et al. usedmagnetic resonance imaging and histochemical staining demonstrated thatdose was the most important variable in maximizing the distribution ofparticles in tissue. Kroll reports that regardless of whether theinfusion volume was small (2 μl) or moderate (24 μl) or whether theinfusion rate was low (6 μl/h) or high (72 μl/h), increasing theparticles from 5.3 to 26.5 μg resulted in as much as a 5-fold increasein the volume of their distribution in tissue.

Concerning cell-type specificity, it has been previously reported thatAAV is capable of transducing neurons and the present study confirmsthis finding. The fact that expression was so prominent in neuronssuggests that AAV gene therapy vectors employing the CMV promoter areuseful for treatment of neurodegenerative diseases such as Parkinson'sand Alzheimer's disease. No expression was seen in mature glial cells,except in small areas of disturbed tissue where active gliosis waspresent. However, we have previously demonstrated that the AAV-CMV-tkvector is expressed well in glioma cells and, when given in conjunctionwith the prodrug ganciclovir, is effective in treating experimentalgliomas in nude mice. Because the tk “suicide” gene is thought to betoxic to dividing cells, it should pose a risk only to the targetedtumor cells and not to surrounding neurons. Finally, while the CMVpromoter used in this study allows for strong transgene expression inneurons, the choice of cell-type-specific promoters will allow targetingof AAV to other CNS components such as oligodendrocytes and glial calls.

The present study also shows that AAV delivered to brain is containedmostly in the central nervous system. Others have demonstratedretrograde transport of viruses between the two hemispheres of brain andability of viruses to reach spinal cord via circulating cerebral spinalfluid. The appearance of vector in the spleen is curious, and suggests acouple of mechanisms. One is that virus enters the bloodstream duringthe infusion process and circulated through the spleen where it is“scavenged”. If this were the case, however, other tissues that havebeen shown to be inducible by AAV would be expected to also take upvirus. Another possible mechanism could be one exhibited by dendriticcells. These cells found mostly in skin, take up foreign material, enterthe circulation, and concentrate in the spleen where the foreign mattercan exist for long periods of time awaiting further processing ordestruction. In any case, we have found that regardless of the route ofdelivery, including intramuscular, intravenous, and now intracerebral,vector is always detected in the spleen.

In summary, slow intracranial infusion of high doses of AAV vector hasbeen shown to transduce a significant portion or brain in a rodentmodel. AAV may be used to target a myriad of central nervous disorders,including tumors, injury resulting from stroke, and neurodegenerativedisease.

Example 3: Gene Therapy of Parkinson's Disease

Convection-enhanced delivery of AAV vectors carrying the transgeneencoding AADC was shown to restore dopaminergic systems in MPTP-inducedParkinson's disease in monkeys as follows.

Animals

Rhesus monkeys (n=4, 3-5 kg) were chosen as candidates for implantationbased on the evolution of their parkinsonian symptoms. Animals werelesioned by infusing 2.5-3.5 mg of MPTP-HLC through the right internalcarotid artery (referred to as ipsilateral side) followed by 4 I.V.doses of 0.3 mg/kg of MPTP-HCL (referred to as contralateral side) untila stable, overlesioned hemi-parkinsonian syndrome was achieved(Eberling, (1998) Brain Res. 805:259-262). The primate MPTP model isconsidered the gold standard model of evaluation prior to human trials.(Langston (1985) Trends Pharmcol. Sci. 6:375-378). MPTP is it convertedin the CNS to MPP+ by monoamine oxidase B. MPP+ is a potent neurotoxinwhich causes degeneration of the nigral dopaminergic neurons and loss ofthe nigro-striatal dopamine pathway, as seen in Parkinson's disease.MPTP-lesioned animals were clinically evaluated once a week using aclinical rating scale and activity monitoring for 5 months prior tosurgery.

Following MPTP administration, the animals developed clinical signs ofParkinson's disease manifested by general slowness, bradykinesia,rigidity, balance disturbances, and flexed posture. The left arm wasless frequently used than the right in all of the monkeys, and allshowed signs of tremor. Using the clinical rating scale, all of themonkeys had moderate to severe stable parkinsonian scores (23±1.7,23+1.2, 24+1.7, 19+3) during the 5 month post-MPTP period.

Vector Production

1. pAAV-AADC:

A 1.5 kb BamHI/PvuII human AADC cDNA (Fan et al. (1998) Human GeneTherapy 9:2527-2535) was cloned into the AAV expression cassette pV4.1cat BamHI/HindII sites. The expression cassette contains a CMV promoter,a chimeric intron composed of a CMV splice donor and a human β-globinsplice acceptor site, human growth hormone polyadenylation sequence, andflanking AAV ITRs (inverted terminal repeats) (Herzog, R. W., Yang, E.Y., Couto, L. B., Hagstrom, J. M., Elwell, D., Fields, P. A., Burton,M., Bellinger, D. A., Read, M. S., Brinkhous, K. M., Podsakoff, G.,Nichols, T. C., Kurtzman, G. J., and High, K. A. (1999) “Long-termcorrection of hemophilia B by gene transfer of blood coagulation factorIX mediated by adeno-associated viral vector”, Nature Medicine5:56-63.).

2. pAAV-LacZ:

The vector pAAV-LacZ was constructed as follows. The AAV coding regionofpSub201 (Samulski et al. (1987) J. Virol 61:3096-3101), between theXbaI sites, was replaced with EcoRI linkers, resulting in plasmidpAS203. The EcoRI to HindIII fragment of pCMVβ (CLONETECH) was renderedblunt ended and cloned in the Klenow treated EcoRI site of pAS203 toyield pAAV-lacZ.

3. pHLp19:

Plasmid H19 encodes a modified AAV-2 genome designed to enhanced AAVvector production while suppressing the generation of replicationcompetent pseudo-wild type virus. The plasmid contains a P5 promotermoved to a position 3′ of the cap gene and the promoter is replaced by a5′ untranslated region primarily composed of a FLP recombinaserecognition sequence. pH19 was constructed so as to eliminate anyregions of homology between the 3′ and 5′ ends of the AAV genome.Additionally, the seven base pair TATA box of the pH19 P5 promoter wasdestroyed by mutation of that sequence to GGGGGGG.

pH19 was constructed in a several step process using AAV-2 sequencesderived from the AAV-2 provirus, pSM620. pSM620 was digested with SmaIand PvuII, and the 4543 bp, rep and cap gene encoding SmaI fragment wascloned into the SmaI site of pUC119 to produce the 7705 bp plasmid,pUCrepcap. The remaining ITR sequences flanking the rep and cap geneswere then deleted by oligonucleotide-directed mutagenesis using thefollowing oligonucleotides:

145A; (SEQ ID NO: 3) 5′-GCT CGG TAC CCG GGC GGA GGG GTG GAG TCG-3′ 145B;(SEQ ID NO: 4) 5′-TAA TCA TTA ACT ACA GCC CGG GGA TCC TCT-3′

The resulting plasmid, pUCepCapMutated (pUCRCM) (7559 bp) contains theentire AAV-2 genome without any ITR sequence (4389 bp). SrfI sites, inpart introduced by the mutagenic oligonucleotides, flank the rep and capgenes in this construct. The AAV sequences correspond to AAV-2 positions146-4,534.

An Eco47III site was introduced at the 3′ end of the P5 promoter inorder to facilitate excision of the P5 promoter sequences. To do this,pUCRCM was mutagenized with primer P547 (5′-GGT TTG AAC GAG CGC TCG CCATGC-3) (SEQ ID NO:5). The resulting 7559 bp plasmid was calledpUCRCM47III.

The polylinker of pBSIIsk+ was changed by excision of the original withBSSHII and replacement with oligonucleotides blunt 1 and 2. Theresulting plasmid, bluntscript, is 2830 bp in length and the newpolylinker encodes the restriction sites EcoRV, HpaI, SrfI, PmeI, andEco47III. The blunt 1 and 2 sequences are as follows:

blunt 1; (SEQ ID NO: 6)5′-CGC GCC GAT ATC GTT AAC GCC CGG GCG TTT AAA CAG CGC TGG-3′ blunt 2;(SEQ ID NO: 7) 5′-CGC GCC AGC GCT GTT TAA ACG CCC GGG CGT TAA CGATAT CGG-3′

pH1 was constructed by ligating the 4398 bp, rep and cap gene encodingSmaI fragment from pUCRCM into the SmaI site of pBluntscript such thatthe HpaI site was proximal to the rep gene. pH1 is 7228 bp in length.

pH2 is identical to pH1 except that the P5 promoter of pH1 is replacedby the 5′ untranslated region of pGN 1909. To do this, the 329 bpAscI(blunt)-SfiI fragment encoding the 5′ untranslated region frompW1909lacZ was ligated into the 6831 bp SmaI(partial)-SfiI fragment ofpH1 creating pH2. pH2 is 7156 bp in length.

A P5 promoter was added to the 3′ end of pH2 by insertion of the 172 bp,SmaI-Eco43III fragment encoding the p5 promoter from pUCRCM47III intothe Eco47III site in pH2. This fragment was oriented such that thedirection of transcription of all three AAV promoters are the same. Thisconstruct is 7327 bp in length.

The TATA box of the 3′ P5 (AAV-2 positions 255-261, sequence TATTTAA)was eliminated by changing the sequence to GGGGGGG using the mutagenicoligonucleotide 5DIVE2 (5′-TGT GGT CAC GCT GGG GGG GGG GGC CCG AGT GAGCAC G-3′) (SEQ ID NO: 8). The resulting construct, pH19, is 7328 bp inlength.

4. pladeno5:

Pladeno 5 is a plasmid that provides a complete set of adenovirus helperfunctions for AAV vector production when transfected into 293 cells.Essentially, it is composed of the E2A, E4, and VA RNA regions fromadenovirus-2 and a plasmid back bone. The plasmid was constructed asfollows.

pBSIIs/k+ was modified to replace the 637 bp region encoding thepolylinker and alpha complementation cassette with a single EcoRV siteusing oligonucleotide directed mutagenesis and the followingoligonucleotide: 5′-CCG CTA CAG GGC GCG ATA TCA GCT CAC TCA A-3′ (SEQ IDNO:9). A polylinker encoding the restriction sites BamHI, KpnI, SrfI,XbaI, ClaI, Bst11071, SalI, PmeI, and NdeI was then cloned into theEcoRV site (5′-GGA TCC GGT ACC GCC CGG GCT CTA GAA TCG ATG TAT ACG TCGACG TTT AAA CCA TAT G-3) (SEQ ID NO:10).

Adenovirus-2 DNA was digested and restriction fragments encoding the E2Aregion (a 5,335 bp, KpnI-SrfI fragment corresponding to positions22,233-27,568 of the adenovirus-2 genome) and the VA RNAs (a 731 bp,EcoRV-SacII fragment corresponding to positions 10,426-11,157 of theadenovirus-2 genome) were isolated. The E2A fragment was installedbetween the SalI and KpnI sites of the polylinker. An E4 region wasfirst assembled in pBSIIs/k+ by ligating a 13,864 bp, BamHI-AvrIIfragment corresponding to adenovirus-2 positions 21,606-35,470 (encodingthe 5′ end of the gene) and a 462 bp, AvrII and SrfI, digested PCRfragment corresponding to adenovirus-2 positions 35,371-35,833 (encodingthe 3′ end of the gene) between the BamHI and SmaI sites of pBSIIs/k+.The oligonucleotides used to produce the PCR fragment were designed tointroduce a SrfI site at the junction were the E4 promoter and theadenovirus terminal repeat intersect and have the sequences 5′-AGA GGCCCG GGC GTT TTA GGG CGG AGT AAC TTG C-3′ (SEQ ID NO: 11) and 5′-ACA TACCCG CAG GCG TAG AGA C-3′ (SEQ ID NO:12). The intact E4 region wasexcised by cleavage with SrfI and SpeI and the 3,189 bp fragmentcorresponding to adenovirus-2 positions 32,644-35,833 was cloned intothe E2A intermediate between the SrfI and XbaI sites. Finally, the VARNA fragment was inserted into the Bst1107 site after T4polymerase-mediated blunt end modification of the SacII site. The genesin pladeno 5 are arranged such that the 5′ ends of the E2A and E4promoters abut, causing the regions to transcribe away from each otherin opposite directions. The VA RNA genes, which are located at the threeprime end of the E4 gene, transcribe towards the E4 gene. The plasmid is11,619 bp in length.

AAV Vector Production

The HEK 293 cell line (Graham, F. L., Smiley, J., Russel, W. C., andNaiva, R. (1977) Characteristics of a human cell line transformed by DNAfrom human adenovirus type 5. J. Gen. Virol. 36:59-72.) was cultured incomplete DMEM (BioWhittaker) containing 4.5 g/liter glucose, 10%heat-inactivated fetal calf serum (FCS), and 2 mM glutamine at 37° C. in5% CO₂ in air. Forty T225 flasks were seeded with 2.5×10⁶ cells each andgrown for three days prior to transfection to 70-80% confluency(approximately 1.5×10⁷ cells per flask).

The transfection and purification methods described by Matsushita et al(Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G., Villarreal,L., Kurtzman, G. J., Iwaki, Y., and Colosi, P. (1998) “Adeno-associatedvirus vectors can be efficiently produced without helper virus,” GeneTherapy 5:938-945) were employed for AAV vector production, with minormodifications. The vector production process involved co-transfection ofHEK 293 cells with 20 μg of each of the following three plasmids perflask: the AAV-AADC plasmid, the AAV helper plasmid (pHLP19, containingthe AAV rep and cap genes), and the adenovirus helper plasmid(pladeno-5, previously known as pVAE2AE4-2 (4) and composed of the E2A,E4, and VA RNA genes derived from purified adenovirus-2), using thecalcium phosphate method (Wigler, M. et al. (1980) Transformation ofmammalian cells with an amplifiable dominant-acting gene. Proc. Natl.Acad Sci. USA 77:3567-3570) for a period of 6 hrs. After transfection,the media was replaced and the cells were harvested 3 days later. Thecell pellets were then subjected to 3 cycles of freeze-thaw lysis(alternating between dry ice-ethanol and 37° C. baths with intermittentvortexing). The cell debris was removed by centrifugation (10,000 g for15 min). The supernatant was centrifuged a second time to remove anyremaining turbidity and subsequently treated with Benzonase® (200 u/ml)at 37° C. for 1 hr in order to reduce contaminating cellular DNA.Following incubation, the supernatant was made 25 mM in CaCl₂, and wasplaced on ice for 1 hr. The resulting precipitate was removed bycentrifugation (10,000 g for 15 min.) and discarded. The supernatant wasthen made 10% in PEG(8000), and was placed on ice for 3 hrs. Theprecipitate was collected by centrifugation (3000 g for 30 min) andresuspended in 4 ml of 50 mM NaHEPES, 0.15M NaCl, 25 mM EDTA (pH 8.0)per 20 T225 flasks. Solid CsCl was added to produce a density of 1.4g/ml and the sample was centrifuged at 150,000 g for 24 hrs in a BeckmanTI70 rotor. AAV-containing fractions were pooled, adjusted to a densityof 1.4 g/ml CsCl, and centrifuged at 350,000 g for 16 hrs in a BeckmanNVT65 rotor. The fractions containing AAV were then concentrated anddiafiltered against excipient buffer (5% sorbitol in PBS). The titer ofthe purified AAV-AADC vector was determined using quantitative dot blotanalysis and vector stocks were stored at −80° C.

Viral Infusion

In the surgery room, a sterile field was created to prepare the infusionsystem. Infusion cannulae were flushed with saline to assess theintegrity between the needle and tubing interface. Sterile infusioncannulae and loading lines were connected using the appropriate fittingswith extreme caution taken to prevent the collection of air bubbles inthe system. Non-sterile oil infusion lines were prepared as previouslydescribed and 1 ml gas tight Hamilton syringes filled with oil wereattached to a Harvard infusion pump. Six infusion cannulae were fittedonto microdialysis holders (3 cannulae per holder) and mounted onto astereotactic tower. Following the union of the oil and loading lines,the needle cannulae were primed with AAV and the infusion systemtransferred to the surgery table. Initial infusion rates were set at 0.1pl/min., the lines visually inspected to ensure a smooth flow of fluidthrough the system, and the cannulae manually lowered to their targetsites. A final visual inspection was performed to check for any airbubbles in the infusion system.

The cannula system consisted of three components: (i) a sterile infusioncannula; (ii) a sterile loading line housing AAV-AADC or AAV-LacZ(control); and (iii) a non-sterile infusion line containing olive oil.Preparation of each line is described here briefly. The infusion cannulaconsisted of 27 G needles (outer diameter, 0.03″; inner diameter, 0.06″;Terumo Corp., Elkton, Md.) fitted with fused silica (outer diameter,0.016″, inner diameter, 0.008″; Polymicro Technologies, Phoenix, Ariz.),and placed in Teflon tubing (0.03″ ID, Upchurch Scientific, Seattle,Wash.) such that the distal tip of the silica extended approximately 15mm out of the tubing. The needle was secured into the tubing usingsuperglue and the system was checked for leaks prior to use. At theproximal end of the tubing, a Tefzel fitting and ferrule were attachedto connect the adjacent loading line.

Loading and infusion lines consisted of 50 cm sections of Teflon tubing(outer diameter, 0.062″; inner diameter, 0.03″) fitted with Tefzel 1/16″ ferrules, unions, and male Luer-lock adapters (Upchurch Scientific,Oak Harbor, Wash.) at the distal ends. The sterile loading linesaccommodated up to a 1000 ml volume and were primed with saline prior touse.

The animals were initially sedated with Ketamine (Ketaset; 10 mg/kg,i.m.), intubated and prepped for surgery. A venous line was establishedusing a 22 gauge catheter positioned in the cephalic or saphenous veinto deliver isotonic fluids at 5-10 ml/kg/hr. Isoflurane (Aerrane, OmedaPPD Inc., Liberty, N.J.) was delivered at 1-3% until the animalmaintained a stable plane of anesthesia. The head was placed in an MRIcompatible stereotactic frame according to pre-set values attainedduring a baseline MRI scan. The animal was instrumented withsubcutaneous electrocardiogram electrodes, a rectal probe and the bodycovered with circulating water blankets to maintain a core temperatureof 36-380 C. Electrocardiogram and heart rate (using the Silogic ECG-60,Stewartstown, Pa.) and body temperature were continuously monitoredduring the procedure. The head was prepped with Betadine and 70%ethanol, a sterile field was created and a midline incision performedthrough the skin, muscle and fascia using electrocautery (SurgistatElectrosurgery, Valleylab Inc., Boulder, Colo.).

Gentle retraction of fascia and muscle allowed for cranial exposure overcortical entry sites. A unilateral craniotomy was performed using aDremel dental drill to expose a 3 cm×2 cm area of dura mater above thetarget sites. Multiple needle cannulae attached to a holder werestereotactically guided to striatal target sites. Surgical parametersfor unilateral infusion of AAV into the hemisphere ipsilateral to ICAMPTP infusion are summarized in Table 2.

TABLE 2 Surgical Parameters for AAV infusion Target Sites Striatum (2caudate, 4 putamen) Hemisphere right side (ipsilateral to ICA MPTPinfusion) Infusion Volume 30 μl/site Infusion Rates 0.1 μl/min (60 min)0.2 μl/min (60 min) 0.4 μl/min (30 min) Virus AAV-AADC; 2.1 × 10e12particles/ml; Lot no 176.12; 200 μl/vial Control Article AAV-LacZ; 9.2 ×10e11 particles/ml; Lot no. 176.126; 200 μl/vial

Approximately fifteen minutes following infusion, the cannulae assemblywas raised at a rate of 1 mm/min. until it was out of the cortex. Thecortex was rinsed with saline, the bone margins trimmed with ronguersand the wound site closed in anatomical layers. Analgesics (Numorphan,1M) and antibiotics (Flocillin, 1M) were administered as part of thesurgical protocol. Animals were monitored for full recovery fromanesthesia, placed in their home cages and clinically observed (2×/day)for approximately five days following surgery. Total neurosurgery timewas 4.5 hours per animal.

Following intrastriatal AAV administration, animals were assessed forany signs of abnormal behavior. Animals were observed and rated by theveterinary technicians twice a day using clinical observation forms. Allmonkeys recovered from the surgery within 2 hours and were able tomaintain themselves, including feeding and grooming. There were no signsof any adverse effects during the entire 8-week post-surgical period.

Magnetic Resonance Imaging

Visualization of the target site is crucial for the precise placement ofcells within the caudate nucleus or putamen. Stereotactic procedurescombined with MRI were used in order to accurately place the cannulawithin the desired targeted structures. All animals were scanned beforesurgery to generate accurate stereotactic coordinates of the targetimplant sites for each individual animal. The same fiducial markers thatare used for PET scanning were placed on the frame for co-registrationof MRI and PET images. Briefly, during the scanning procedure, theanimals were sedated using a mixture of ketamine (Ketaset, 7 mg/kg, im)and xylazine (Rompun, 3 mg/kg, im). The animals were placed in an MRIcompatible stereotactic frame, earbar and eyebar measurements wererecorded, and an IV line was established. Sixty coronal images (1 mm)and 15 sagittal images (3 mm) were taken using a GE Signa 1.5 Teslamachine. Magnetic resonance images were T1-weighted and obtained inthree planes using a spoil grass sequence with a repetition time(TR)=700 ms, an echo time (TE)=20 ms and a flip angle of 30′). The fieldof view was 15 cm, with a 192 matrix and a 2 NEX (number of averages persignal information). Baseline scanning time was approximately 20minutes. Rostro-caudal and medio-lateral distribution of a targetedstructure (e.g., caudate nucleus) was determined using the coronal MRimages. Surgical coordinates were determined from magnified coronalimages (1.5×) of the caudate nucleus and putamen.

Positron Emission Tomography (PET)

All 4 animals received 2 PET scans, a baseline scan followingestablishment of the MPTP lesion, and a second scan 7-8 weeks afterinfusion with either AAV-AADC or AAV-LacZ. Prior to PET, each animalunderwent magnetic resonance (MR) imaging using a 1.5 T magnet and astereotaxic frame which permitted coregistration between PET and MR datasets through the use of external fiducial markers. The PET studies wereperformed on the PET-600 system, a singleslice tomograph with aresolution of 2.6 mm in-plane and an adjustable axial resolution whichwas increased from 6 mm to 3 mm for the current study by decreasing theshielding gap. The characteristics of this tomograph have been describedpreviously (Budinger et al. (1991) Nucl. Med Biol. 23(6):659-667; Valk,(1990) Radiology 176(3):783-790). The monkeys were intubated andanesthetized with isoflurane, placed in a stereotaxic frame andpositioned in the PET scanner so as to image a coronal brain slicepassing through the striatum. Monkeys were positioned in the same wayfor each study using the anterior-posterior scales on the sterootaxicframe and a laser light connected to the tomograph. After beingpositioned in the scanner, a 5 min transmission scan was obtained inorder to correct for photon attenuation, and to check the positioning ofthe animal. The monkeys were then injected with 10-15 mCi of the AADCtracer, 6-[¹⁸F]fluro-L-m-tyrosine (FMT) and imaging began. Imagingcontinued for 60 min, at which time the monkey was repositioned so as toimage a second slice 6 mm caudal to the first.

The PET and MR datasets were co-registered and regions of interest (ROs)were drawn for the striatum in the contralateral hemisphere (the sideopposite to ICA MPTP infusion) on PET data collected at 50 to 60 min(slice 1) and from 65 to 75 min (slice 2) with reference to the MR.Mirror images of the ROs were created in the ipsilateral hemisphere(side of MPTP infusion) and radioactivity counts (cm2/sec) weredetermined for each ROI. Striatal counts were averaged over the twoslices for each study. FMT uptake asymmetry ratios were calculated foreach animal at each time point by subtracting the counts for theipsilateral (lesioned) striatum from the counts for the contralateral(un-lesioned) striatum and dividing by the average counts for theipsilateral and contralateral striata. In order to reduce between animalvariability in asymmetry ratios, a change score was calculated bysubtracting the asymmetry ratio from the second PET study from theasymmetry ratio for the baseline study for each animal. Unpaired t-testswere used to compare the change in pet asymmetry ratios for the AAV-AADCand AAV-LacZ monkeys.

As expected, all 4 monkeys showed greater FMT uptake in thecontralateral than in the ipsilateral striatum at baseline, which showednegligible uptake. At the time of the second PET study, the AAV-AADCtreated monkeys showed increased FMT uptake in the ipsilateral striatum,while the AAV-LacZ treated animals showed no change from baseline. (FIG.7). The change in FMT uptake asymmetry from baseline to the second PETstudy was significantly (p<0.01) greater for the AAV-AADC monkeys, whichshowed little asymmetry at the time of the second study, than for theAAV-LacZ monkeys, which showed greater contralateral FMT uptake at bothtime points. (FIG. 8)

Necropsy

Animals were deeply anesthetized with sodium pentobarbital (25 mg/kgi.v.) and sacrificed 8-9 weeks following AAV administration and one weekfollowing postsurgical PET scans. On the day of sacrifice, blood sampleswere taken, and the animals were treated with L-dopa/carbidopapreparation (Sinemet 250/25). Plasma and cervical CSF were collected andat the time of necropsy. The brains were removed 30-45 minutes followingthe Sinemet administration, placed in the brain matrix and sectionedcoronally into 3-6 mm slices. One 3 mm thick striatal brain slice fromeach monkey was immediately frozen in −70° C. isopentane and storedfrozen for biochemical analysis. The remaining 6 mm thick slices werepost-fixed in formalin for 72 hours, washed in PBS for 12 hrs andadjusted in ascending sucrose gradient (10-20-30%) and frozen.

Histological Analysis

The formalin-fixed brain slices were cut into 30 μm thick coronalsections in a cryostat. Frozen sections were collected in seriesstarting at the level of the rostral tip of the caudate nucleus all theway caudally to the level of the substantia nigra. Each section wassaved and kept in antifreeze solution at 70° C. Serial sections werestained for tyrosine hydroxylase (TH), dopa decarboxylase (DDC) orB-galactosidese (B-gal) immunorectivity (IR). Every 12th section waswashed in phosphate buffered saline (PBS) and incubated in 3% H2O2 for20 min to block the endogenous peroxidase activity. After washing inPBS, the sections were incubated in blocking solution (10% normal horseserum for TH or 10% normal goat serum for DDC and B-gal and 0.1%Triton-X 100 in PBS) for 30 min, followed by incubation in primaryantibody solution-TH (mouse monoclonal, Chemicon, 1: 1000), DDC (rabbitpolygonal, Chemicon, 1:2000) or B-gal (rabbit polygonal, Cortex Blochem,1:5000) for 24 h. The sections were then incubated for 1 h inbiotinylated anti-mouse IgG secondary antibody for TH or anti-rabbit IgGsecondary antibody for DDC and B-gal (Vector Labs, 1:300). The antibodybinding was visualized with streptavidin horseradish peroxidase (VectorLabs, 1:300) and DAB chromogen with nickel (Vector Labs). Sections werethen coverslipped and examined under a light microscope.

Following tissue punching the fresh-frozen blocks were sectioned at 20um. Sections were stained with H&E and for DDC-IR. (fig xx)

Quantitative estimates of the total number of AAV-infected cells withinthe caudate nucleus, putamen and globus pallidus were determined byusing an optical dissector procedure. The optical dissector systemconsisted of a computer assisted image analysis system, including anLeitz Otholux 11 microscope hard-coupled to a Prior H128computer-controlled x-y-z motorized stage, a high sensitivity Sony 3CCDvideo camera system (Sony, Japan) and a Macintosh G-3 computer. Allanalyses were performed using NeuroZoom software (La Jolla, Calif.).Prior to each series of measurements, the instrument was calibrated. Theregion of positive neurons in the caudate, putamen and globus palliduswas outlined at low magnification (2.5× objective). Because of thediffuse presence of AAV-infected cells within the striatum, 1% of theoutlined region was measured with a systematic random design ofdissector counting frames (1 505 1IM2) using a 63× plan-neofluarimmersion objective with a 0.95 numerical aperture. Based on pilotexperiments at least four sections equally spaced were sampled. By usingthe dissector principle, up to 200 AADC positive neurons were sampled byoptical scanning by using uniform, systematic and random designprocedures for all measurements. The average thickness of the sectionswas measured at 23 microns. Once the top of the section was in focus,the z-plane was lowered a 1-2 gm. Counts were than made while focusingdown through three 5 lim-thick dissectors. Care was taken to ensure thatthe bottom forbidden plane was never included in the analysis. Thevolumes of the structures were calculated according to the procedure ofthe Cavalleri (Cavalieri, 1966). The total number of positive cells inthe examined structures was calculated by using the formula N=Nv×Vs,where Nv is the numerical density and Vs is the volume of the structure.

TH-IR staining revealed robust reduction of the nigrostriatal fibers andcell bodies in the substantia nigra on the ipsilateral side in all ofthe monkeys. The contralateral side showed variable reductions of TH andAADC-IR in the striatum and the substantia nigra. DDC-IR paralleledTH-IR only in the monkeys treated with AAV-LacZ. The AAVAADC-treatedmonkeys showed robust AADC staining on the ipsilateral side thatexceeded staining seen on the contralateral side. A high density ofAADC-IR cells was seen throughout 80% of the striatum and 100% of theglobus pallidus in one of the AAV-AADC treated animals. Stereologicalanalysis revealed 18,384 cells per mm³ in the putamen, 15,126 cells permm³ in the caudate and 9,511 cells per mm³ in the globus paillidus. Thetotal number of AAV-infected cells was estimated to be at least 16×10′cells. In the other AAV-AADC treated monkey, AADC-IR cells were found inover 60% of the ipsilateral striatum, with 7,515 cells per mm³ in thecaudate and 15,352 cells per mm³ in the putamen and 3,850 cells per mm³in the globus pallidus. No AADC cells were found in the contralateralstriatum. The AAV/LacZ-treated monkeys did not show AADC-IR in eitherthe ipsilateral or contralateral striata.

Cells infected with AAV appeared to have neuronal morphology. Theaverage diameter of the infected cells was 9±2.3 μm in the putamen and14.6±9 μm. Many Lac-Z and AADC cells had a typical medium spiny neuronmorphology. AAV-infected cells were positive for the neuronal marker,Neu-N. In the AAV-AADC-treated monkeys, one out of 4-6Neu-N-positivecells was AADC-positive in the caudate and putamen, and one out of 3-4Neu-N-positive cells was AADDC positive in the globus pallidus. None ofthe AAV-infected cells in the Lac-Z or AADC-treated monkeys wereGFAP-positive.

Areas adjacent to cannula tracts were stained with Nissi and H&Estaining. No signs of cytotoxicity were observed. No perivascularcuffing was observed, regardless of the distance from the cannula. Therewere no signs of neuronal cell reduction close to the infusion site whencompared to the contralateral side using Neu-N immunostaining.GFAP-immunostaining failed to detect any abnormal glia reaction withinthe AAV-treated striatum.

Biochemical Analysis

Brain regions were removed from fresh frozen blocks using a micropuncherin order to evaluate tissue levels of L-dopa and dopamine metabolitesand the activity of AADC and the presence of the AAV-vector. Brainregions included striatum and cortex as shown in fig ??.

Frozen micropunches were collected, and homogenized by ultrasonicprocessing in 300 μl of 0.1 M perchloric acid (Fisher Scientific)containing 1% ethanol, and 0.02% EDTA (Fisher Scientific). 50 μl of thehomogenate was removed for protein analysis (BCA Protein Assay KitPierce #23225), and the remainder centrifuged in a mirocentrifuge for1.5 minutes at maximum speed. 30 to 50 pl of the homogenate was used forcatecholamine analysis by HPLC using an Ultrasphere C-18 ion pair, 5 p,4.6×250 mm column (Beckman 235329); a Waters 717plus autosampler at 4°C., Waters 51 0 pump at 0.9 mvmin, and amperometric electrochemicaldetector (Decade) set at Eox. 0.82V. The column and detector cell wereset at 31° C. The mobile phase contained 2 L HPLC grade water, 2.2 g1-heptanesulfonic acid, sodium salt (Fisher Scientific), 0.1 7 g EDTA,12 ml triethylamine (Fisher Scientific), pH adjusted to 2.5 with=−8 ml85% phosphoric acid (Fisher Scientific), and 60 ml acetonirile (J. T.Baker). The detector output was recorded and analyzed with the WatersMillennium 32 Chromatography Manager.

AADC Analysis

AADC activity was determined by an adaptation of the method of Nagatsuet al (1979) Anal. Biochem. 100:160-165. Briefly, tissue (10 mg/ml) washomogenized in 50 mM phosphate buffer (pH 7.4) containing 0.04 mMpyrixyl phosphate (a AADC cofactor) and 0.2 mM pargyline. Samples werepre-incubated at 37° C. for 5 minutes and the reaction was initiated bythe addition of L-dopa (final concentration: 100 μM). Incubations werecarried out for 20 minutes and the reaction stopped by the addition of0.02 ml concentrated perchloric acid. After centrifugation, thesupernatant dopamine concentration was determined using HPLC withelectrochemical detection. (see, e.g., Boomsa et al. (1988) Clin. Chem.Acta 178-59-69). Protein concentration in the tissue pellet wasdetermined using the BCA Protein Assay Kit (Pierce #23225). Results areexpressed as nM/hr/mg of protein. Frozen tissue punches were processedaccording to the procedure of Irwin et al.

Cortical regions of all monkeys showed variable levels of L-dopa,however, they were consistent within each monkey. As expected, there wasno decarboxylation of L-dopa to dopamine within the cortex, however, inthe striatum on the side contralateral to MPTP administration, L-dopawas converted to dopamine and further metabolized to HVA. In theMPTP-treated striatum of the AAV-Lac-Z monkeys, L-dopa was not convertedto dopamine, nor was it metabolized to HVA. Tissue levels of L-dopa alsoremained at the same levels as in the cortex in AAV-Lac-Z treatedmonkeys. In the MPTP-treated striatum of AAV-AADC-treated monkeys,L-dopa was converted to dopamine and HVA and tissue levels of L-dopa inthis region were reduced.

AADC activity was very low in the cortical regions and in theMPTP-treated striatum of AAV-LacZ-treated monkeys. L-dopa was convertedto dopamine in the contralateral striatum, suggesting high levels ofAADC activity. The tissue punches from MPTP-treated striatum of AAV-AADCinfected monkeys contained extremely high dopamine levels with onlytraces of L-dopa left.

These results demonstrate that the combination of infused AAV-AADCvector and systemic L-dopa is a promising therapy for the treatment ofPD.

Thus, the invention provides a novel and efficient treatment method forCNS disorders, such as Parkinson's Disease. In addition, the inventionalso provides methods for determining dopamine activity in vivo.

What is claimed is:
 1. A method for delivering recombinant AAV virions to a subject, comprising administering via convection-enhanced delivery (CED) said rAAV virions into the CNS of the subject, wherein said rAAV virions comprise a nucleic acid sequence encoding a therapeutic polypeptide.
 2. The method of claim 1, wherein the administering is done with an osmotic pump.
 3. The method of claim 1, wherein the administering is done with an infusion pump.
 4. The method of claim 1, wherein the nucleic acid sequence encodes an aromatic-amino-acid decarboxylase (AADC).
 5. The method of claim 1 wherein the subject is a human.
 6. The method of claim 1, wherein the rAAV virions are administered into the striatum.
 7. A method for delivering recombinant AAV virions to a subject having a CNS disorder, comprising administering via convection-enhanced delivery (CED) said virions into the CNS of the subject, wherein said virions comprise a nucleic acid sequence encoding a therapeutic polypeptide.
 8. The method of claim 7 wherein the CNS disorder is Parkinson's disease, the rAAV virions are administered into the striatum and wherein the nucleic acid sequence encodes AADC
 9. A method for treating a neurodegenerative disease in a subject, said method comprising: (a) providing a preparation comprising recombinant adeno-associated virus (rAAV) virions, wherein said virions comprise a nucleic acid sequence that is expressible in transduced cells to provide a therapeutic effect in the subject; and (b) delivering the preparation to the CNS of the subject using convection-enhanced delivery (CED), wherein said virions transduce neural cells and the nucleic acid sequence is expressed to provide a therapeutic effect in the subject suitable for treating said neurodegenerative disease.
 10. The method of claim 9, wherein the neurodegenerative disease is Parkinson's disease.
 11. The method of claim 9, wherein the nucleic acid sequence expressible in transduced cells encodes AADC or functional fragment thereof.
 12. The method of claim 9, further comprising administering to the subject at least one additional therapeutic compound.
 13. The method of claim 12 wherein the at least one additional therapeutic compound is L-dopa.
 14. The method of claim 13, further comprising administering L-dopa and, optionally, carbidopa to the subject.
 15. A method of determining levels of dopamine activity in the brain of subject comprising; (a) administering a labeled tracer to the subject, wherein binding of the tracer to a cell is indicative of dopamine activity; and (b) imaging the subject's brain to determine the number of cells which bind the labeled tracer, thereby determining levels of dopamine activity in the subject's brain.
 16. The method of claim 15, wherein the labeled tracer is 6-[¹⁸F]-fluoro-L-m-tryosine (¹⁸F-FMT).
 17. The method of claim 15, wherein the imaging is positron emission tomograph (PET) imaging. 